Efficacy of a combined protocol for re-insemination of open cows after early pregnancy diagnosis using ultrasonography and its effect on fertility.

The objective of the present field study was to establish the beneficial effects of re-insemination of non-pregnant cows using ultrasonography 20 to 23 days after the artificial insemination. A total of 245 Japanese Black cows were artificially inseminated and early pregnancy diagnosis (EPD) was performed on 92 cows 20 days after insemination, using ultrasonography. The remaining 153 cows were considered as negative controls in which routine rectal palpation was performed for pregnancy diagnosis 45-50 days post-insemination. EPD revealed that eleven of the 92 cows (12%) were infertile due to ovarian abnormalities and were thus excluded from the rest of the study. Forty-eight (59%) of the remaining 81 cows were diagnosed as pregnant, while the other 33 (41%) were diagnosed as non-pregnant. Of these non-pregnant cows, 17 of them received a dose of an analogue of the gonadotropin-releasing hormone (GnRH analogue) and were then timed-inseminated, while the other 16 were observed for estrus signs, and 13 of them (81%) were artificially inseminated. Rates of conception were 35% and 38% in the GnRH and the artificially inseminated groups, respectively (P>0.05). Total pregnancy rate for the EPD group increased significantly (74%) (P<0.01) when compared to the control cows (54%) within the same period. In conclusion, our field study demonstrated that re-insemination of non-pregnant cows following EPD is highly efficacious not only in improving the rate of fertility via reducing inter-insemination and inter-calving intervals, but also aids in the early detection of ovarian disorders.


Introduction
On modern cattle farms, any delay in the conception of non-pregnant cows is deemed unacceptable financial loss (Oltenacu et al., 1990). Several methods of early pregnancy diagnosis (EPD) have been employed to detect the non-pregnant cows for the purpose of re-insemination. Such methods include rectal palpation, starting from day 35 post insemination (Thompson et al., 1995), hormonal assay using milk, blood, or feces to detect the progesterone levels (Katagiri et al., 2002, Faustini et al., 2007Isobe et al., 2005), ultrasonography to detect the embryo(s) starting from day 26 (Ramano et al., 2006) and some other laboratory methods; such as using radioimmunoassay of a pregnancy-specific protein in the plasma and early conception factor (ECF) test (Humblot et al., 1988;Cordoba et al., 2001) or simply the non-return to estrus method. However, all these methods have few disadvantages regarding the days at which diagnosis is performed; this is because with those methods, the non-pregnant cows cannot be diagnosed early enough to be treated for infertility if they do not exhibit the typical signs of estrus. In such cases they should be synchronized for estrus as soon as possible (Lucy, 2005) or just left to wait until the next natural estrus; which implies wasting of valuable resources such as time, money, and effort (Oltenacu et al., 1990). Therefore, early detection of non-pregnant cows after insemination is considered an essential process that enables us to make a right decision at a proper time, especially when the early detection method is combined with the re-insemination program. Thus, the objective of the present field study was to establish the advantages of re-insemination of non-pregnant cows diagnosed using ultrasonography as early as 20 to 23 days post insemination.

Animals
Two hundred and forty five (245) Japanese Black cows (raised in a cattle farm in Sendai city/Japan) were used for this study. Feeding of animals in this farm was according to the standard Japanese Animal Feeding Programs, AFFRC (2004).

Method of Pregnancy Diagnosis
EPD was performed according to the procedure described by Gaja et al. (2009), which involves monitoring the cross sectional areas of corpora lutea (CLc-s area). Ovaries of 92 cows were scanned using real-time ultrasonography (Tringa linear, Esaotepie medical, Netherlands), equipped with a 5 MH linear transducer. Open (non-pregnant) cows were randomly divided into two groups: Group1 (n=17): Cows received an analogue of the gonadotropin-releasing hormone (GnRH analogue) (10 μg, Buserelin, Teikoku Zoki Co. Ltd, Japan) and were inseminated artificially, using timed artificial insemination (TAI) technique 16-20 hours post GnRH injection. (N.B.: At the time of injection, all the cows in this group had dominant follicles, on their ovaries, of more than 10 mm diameter). Group2 (n=16): Cows were inseminated artificially according to the a.m.-p.m. rule after the detection of estrus. Ultrasonography scanning was performed by the same expert person; avoiding an invasive manipulation of genitalia. The cross-sectional areas (mm 2 ) of CLs (CL c-s areas) were calculated using the following formula: CL c-s area (elliptical area) = π× (diameter a/2) × (diameter b/2) {Where (a) and (b) are the long and the short diameter of the CL, respectively}. Regression rate of the CL was calculated using the following formula: 100-(day X 1 CL c-s area/ day X 2 CL c-s area) × 100. {Where X 1 and X 2 represent the 1 st and 2 nd scanning days, respectively}. The remaining 153 cows were diagnosed for pregnancy using the routine farm practice (rectal palpation on day's 45 -50 post insemination).

Statistical Analysis
For statistical analysis, significant differences between the pregnant and non-pregnant cows were determined using a chi-square test. A P-value less than 0.05 were considered statistically significant.
Results All cows (n=245) exhibited estrus and were inseminated artificially. 153 cows were considered as negative controls, in which routine rectal palpation was performed for pregnancy diagnosis 45-50 days post-insemination.EPD was performed on 92 cows 20 days after insemination, using ultrasonography. During the first ultrasonographic examination, ovarian disorders were discovered in 11 cows (12%). These cows were excluded from the remaining part of the experiment. On the second ultrasonographic examination of the remaining 81 apparently-healthy cows, which was performed within days 20 to 23, the results showed that48 (59%) of the remaining 81 cows were diagnosed as positively pregnant. On the other hand, 33 (41%) of the remaining 81 cows were diagnosed as non-pregnant. The 33 non-pregnant cows were further divided into two groups designated as group 1 and group 2. All cows in group1 (n=17) received GnRH analogue and were inseminated using TAI 16-20 hours post GnRH injection. All cows in group 2 (n=16) were observed for estrus, and 13 of them (81%) were artificially inseminated (AI). Conception rate (which denotes the number of positive pregnancies) was 35% (6 out of 17) in the GnRH +T AI group (group 1) and 38% (6 out of 16) in AI group (group 2), (P>0.05). Total pregnancy rate (1 st and 2 nd inseminations of treated cows), increased (60/81, 74%) when compared to the pregnancy rate in the 1 st inseminated cows of treatment (48/81, 59%) and the cows in the control group (82/153, 54%) within the same period (P<0.01).
Discussion This study is a field application of our previous research on EPD in Japanese Black cows (Gaja et al., 2009;Gaja et al., 2012), in which we found that encouraging results can be obtained without any delay using transrectal ultrasonographic imaging of corpora lutea cross-sectional areas (CLs c-s areas), a protocol which is followed by double verification on days 13 to 16 and 20 to 23 post insemination. Experimental procedures employed in the present study gave us a unique opportunity for an early diagnosis of pregnancy, especially for the non-conceived cows. Thus, these cows could be re-inseminated as early as possible after being diagnosed as non-pregnant without any undue wasting of more open-days. In our recent study, (Gaja et al., 2012), we determined the relation between the regression rate of CL c-s area between 13 to 16 and 20 to 23 days and the pregnancy status, considering that the first examination (days 13 to 16 post insemination) is 100%. For more a precise and feasible technique which could be efficiently applied in the field to diagnose pregnancy in cows as early as possible without wasting time by waiting for the next estrus, certain values for the rate of CL c-s area`s change between first and second examinations for pregnancy should be proposed. Inseminated cows can be assertively concluded as non-pregnant if the regression of their CL exceeded 25% from their initial value using ultrasonographic examinations. If the regression is less than 10%, cows can be considered to be most likely conceived, while those which show regression rates between 10-25% are advised to be re-